Th0 cells are Th cells that are capable of making both IL-2 and IL-4. Given that Th0 cells possess all of the cellular machinery required to produce both IL-2 and IL-4, they provide a powerful system for studying the intracellular signals that regulate the production of these two important lymphokines. Lymphokine production in Th0 cells can be driven towards the production of these two important lymphokines. Lymphokine production in Th0 cells can be driven towards the production of only IL-2 or only IL-4 by 1) elevating intracellular cAMP, 2) using different APC, and 3) treating the cells with anti-Thy-1 mAb. this proposal tests the hypothesis that Th0 cells use separate and distinct intracellular signalling pathways for the production of the lymphokines IL-2 and IL-4. Aim 1. We will expand our panel of Th0 hybridomas and APC to be used in the intracellular signalling experiments, and determine whether the mode of delivery of the primary stimulus affects the response to these modulators. We also will identify stimulation conditions which minimize the effect of T cell-derived costimulation, and thus T cell density, on lymphokine production. As an additional control for T cell density, lymphokine mRNA expression will be measured from cells stimulated under the conditions used for the signalling studies. Aim 2. In some Th0 cells, cAMP selectivity inhibits the production of IL-2, while enhancing IL-4 production; in other Th0 cells, both IL-2 and IL-4 are inhibited. These differences may derive from differences in cAMP regulation and/or the activation of protein kinase A (PKA). We will examine basal and stimulated levels of cAMP and the expression and function of the 2 major isozymes of PKA. The pattern of PKA-mediated phosphorylation also will be examined, using high resolution 2-D gel electrophoresis. Aim 3. The Th0 hybridomas will be used to analyze the intracellular signalling pathways involved in the production of IL-2 and IL-4. First, it will be determined whether the 3 modulators examined here (cAMP, different APC, and anti-Thy-1 mAb) affect IL-2 or IL-4 mRNA stabilization or whether they act by transcriptional control. Second, tyrosine phosphorylation will be examined. It will be determined whether inhibitors of protein tyrosine kinases or phosphatases selectively alter lymphokine production patterns in any or all Th0 cells. The kinases p56lck and p59fyn will be specifically inhibited by introduction of functionally inactive forms of the enzyme using retroviral vectors. In addition, 2-D gel electrophoresis will be used to analyze patterns of tyrosine phosphorylation induced in Th0 cells incubated with cAMP, different APC, or anti-Thy-1 mAbs. Third, activation of the PLC/PKC pathway will be explored. PKC inhibitors will be used to attempt to selectively affect IL-2 and IL-4 production. Finally, the effect of the modulators (cAMP, different APC, and anti-Thy-1 mAbs) on intracellular [Ca2+], PIP2 hydrolysis, and PKC activation will be studied. These experiments should provide the first data on the signalling pathways that selectively lead to the production of IL-2 and IL-4.